Abstract
The yeast Cch1/Mid1 Ca2+ channel is equivalent to animal voltage-gated Ca2+ channels and activated in cells incubated in low Ca2+ medium. We herein investigated the third subunit, Ecm7, under the same cell culture conditions. The deletion of ECM7 slightly lowered Ca2+ influx activity in the CNB1+ background, in which calcineurin potentially dephosphorylates Cch1, but markedly lowered this activity in the cnb1Δ background. The deletion of the C-terminal cytoplasmic region of Ecm7 also reduced Ca2+ influx activity. We identified a novel Cch1-interacting protein, Scs2, which is known as a cortical endoplasmic reticulum membrane protein. The deletion of SCS2 did not affect Ca2+ influx activity when calcineurin was inhibited by FK506, but enhanced this activity by 35% when the enzyme was not inhibited. However, this enhancement was canceled by the deletion of ECM7. These results suggest that Cch1/Mid1 is regulated differentially by Ecm7 and Scs2 in a manner that is dependent on the phosphorylation status of Cch1.
Highlights
Ca2+ is a second messenger that induces changes in many fundamental cellular events, including enzyme activation, secretion, and gene expression [1]
In order to study the role of Ecm7, we employed low Ca2+ medium containing 100 μM CaCl2, named SD.Ca100, in which high affinity Ca2+ influx system (HACS) becomes active in α-factor-treated cells [4,7]
Viability assays showed similar results between the ecm7Δ and cch1Δ mutants and wild-type strain (Fig 2B). These results suggest that Ecm7 only negligibly contributes to HACS in cells exposed to α-factor, even in low Ca2+ medium
Summary
Ca2+ is a second messenger that induces changes in many fundamental cellular events, including enzyme activation, secretion, and gene expression [1]. The permeation of Ca2+ through cellular membranes is crucial and strictly regulated by a number of transporters and channels, one of which belongs to the voltage-gated Ca2+ channel (VGCC) superfamily. Regulation of Cch by Ecm and Scs. Animal VGGCs are composed of four subunits, such as the pore-forming α1 subunit and three auxiliary subunits, α2/δ, β, and γ, and are regulated by membrane-located and cytoplasmic proteins [2, 3]. The role of the auxiliary subunits is to modulate the level of expression and voltage dependence of the α1 subunit. The γ subunit generally exerts smaller effects
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